It has now become feasible for cellular biochemists to determine the chemical constituents of a single cell. This breakthrough has been achieved as a result of the discovery of new chemical separation and detection methodologies which are sensitive at the femtomole level and below. Moreover, it is now possible to manipulate a wide variety of cells and even to insert electrochemical probes inside the cores of these materials for in-vivo studies. These tools allow elucidation of the function of single cells, as contrasted to assemblies of cells and are driving new approaches to an understanding of the mechanisms of cellular functions.
Subcellular localization and imaging of molecules can provide a further dimension for monitoring of chemical processes. The imaging of such molecules is extremely difficult. Most biological cells have diameters in the range of 1-10 microns, although a few types of neurons are known to be as large as 500 microns. To provide a reasonable spatial image of a molecule, there should be at least 10 picture elements (pixels) along each dimension of the cell. Submicron molecular imaging requires both spatial resolution below the resolving power of optical systems and molecular specificity to enable detection of species of different atomic weights.
There have been a number of methods developed in order to approach sub-micron imaging. The most sensitive methods involve the detection of specific types of atoms which may also act as tags for a target molecule. X-ray microanalysis, electron-energy-loss spectroscopy, Auger electron spectroscopy and secondary ion mass spectrometry (SIMS) have been pursued in this regard.
For some time it has been known that molecular ions may be desorbed from surfaces bombarded by heavy keV particles. This procedure is known as secondary ion mass spectroscopy (SIMS) and involves the use of an ion source to bombard the sample and a subsequent measurement of the mass of desorbed particles. In its dynamic form, SIMS employs a high energy ion beam that erodes the sample but provides a high level of desorbed particles for detection purposes. In another form, known as "static" SIMS, a considerably smaller dose of incident ions is employed having a beam density sufficiently low so as to avoid sample surface damage (usually less than 10.sup.13 ions per square centimeter). Static SIMS has the advantage that mass spectra of surfaces can be obtained but suffers the disadvantage that only few desorbed particles are available for measurement.
Recently, a new SIMS technique has been developed that is referred to as "time-of-flight" SIMS. This method employs a pulsed ion gun and can be operated in the static SIMS mode because of its high collection efficiency. The time-of-flight of the desorbed species allows derivation of the species' mass. Imaging has been performed in a time-of-flight SIMS system, but submicron spatial resolution for molecular species has not been feasible since there are not sufficient desorbed ions for measurement purposes.
Another spectrometry method has been reported that overcomes some of the sensitivity drawbacks of prior art static SIMS techniques. That procedure is termed "sputter initiated resonance ionization spectrometry" and is further described in U.S. Pat. Nos. 3,987,302; 4,426,576; 4,442,354 and 4,658,135 to Hurst et al. The Hurst et al. system bombards a sample with ions to create a cloud of secondary ions and neutral particles. The cloud is irradiated with a laser pulse that is tuned to ionize the neutral particle species by means of multiphoton resonance ionization. Then, the mass of the ionized particles is determined by a time of flight spectrometry method. The laser irradiation enables the creation of a substantially greater quantity of ionized species for measurement purposes, but requires a 1-2 millimeter diameter focused laser spot. If a more tightly focused spot is employed, much fewer ions are generated, even with the laser assisted ionization.
Still another mass spectrometry method is described by Becker et al in U.S. Pat. No. 4,733,073. In the Becker et al system, a highly focussed laser beam is employed to ionize particles from a sample surface. The laser beam is not tuned to achieve resonance ionization, but achieves ionization action through the beam's high power. Due to the tight focus of the beams, however, the beam interacts with just a small portion of the cloud of particles over the sample's surface, and particle sensitivity suffers.
Concomitant with the development of static SIMS has been the discovery that a liquid metal ion gun (LMIG) can be employed to focus ion beams to a spot size as small as 200 Angstroms in diameter. The LMIG is characterized by an extremely high current density that enables high sensitivity but introduces a number of artifacts due to beam damage when operated in the dynamic mode. The LMIG has recently been commercialized and incorporated into a high transmission reflectron time-of-flight analyzer. By taking advantage of its high detection efficiency and by operating this instrument in the static mode, others have been able to image tripeptides from a conducting substrate with spatial resolutions approaching one micron.
A traditional problem with SIMS and other spectrometry systems that rely upon ion bombardment is the presence of large matrix ionization effects. Both the ionization potential of the target molecule, as well as the electronic properties of the matrix surrounding the molecule exponentially influence the probability of ion formation. Furthermore, the number of ions produced during desorption is generally several orders of magnitude smaller than the number of neutral species. Ionization effects have thus created severe problems with such spectrometry methods.
Accordingly, it is an object of this invention to provide an atomic and molecular imaging system that exhibits resolution levels in the hundreds of Angstroms.
It is another object of this invention to provide an improved resonance ionization spectrometry system for imaging the presence of molecular species across a surface.
It is still another object of this invention to provide a molecular level imaging system that employs a highly focused ion beam that does not materially change the surface properties of the sample.